A C4N4 Diaminopyrimidine Fluorophore

Article abstract of DOI:10.1002/chem.201900467

A new scaffold for producing efficient organic fluorescent materials was identified: 2,5-diamino-4,6-diarylpyrimidine featuring a C4N4 elemental composition. Single-step installation of two aryl groups at the 4,6-positions of the pyrimidine core delivered

Full text of DOI:10.1002/chem.201900467

DOI: 10.1002/chem.201900467
Communication
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Fluorescence
A C4N4 Diaminopyrimidine Fluorophore
Hidetoshi Noda, Yasuko Asada, Tatsuro Maruyama, Naoki Takizawa, Nobuo N. Noda,
Masakatsu Shibasaki,* and Naoya Kumagai*[a]
though these privileged fluorophores have broadly contributed
Abstract: A new scaffold for producing efficient organic
fluorescent materials was identified: 2,5-diamino-4,6-
to bioimaging applications, the identification of a new fluoro-
genic scaffold would expand the chemical space of biocompat-
diarylpyrimidine featuring a C4N4 elemental composition.
ible fluorescent dyes, covering broadened chemical and photo-
Single-step installation of two aryl groups at the 4,6-posi-
physical properties to meet the growing demand of bioimag-
tions of the pyrimidine core delivered fluorescent organic
ing needs.[4] Herein, we report a new scaffold for organic fluo-
materials in a modular fashion. A range of fluorescent
rescent materials characterized by a unique 2,5-diamino-4,6-di-
compounds with distinct absorption/emission properties
arylpyrimidine with a C4N4 elemental composition (Figure 1).[5]
was readily accessed by changing the aromatic attach-
ments. A generally high absorption coefficient and quan-
tum yield were observed, including C4N4 derivatives that
could fluoresce even in the solid state. The two amino
groups at the 2,5-positions of the pyrimidine were essen-
tial for intense fluorescence with a large Stokes shift,
which was corroborated by structural relaxation to a
p-iminoquinone-like structure in the excited state. Besides
live-cell imaging capabilities, fluorescent labeling of a pro-
tein involved in autophagy elucidated a new protein–pro-
tein interaction, supporting potential utility in bioimaging
applications.
Figure 1. Overview of a 4,6-diarylated 2,5-diaminopyrimidine fluorophore
featuring a central C4N4 unit.
The growing number of potential uses for organic fluorescent
materials in a range of research fields has drawn increased at-
tention to the design and synthesis of emissive organic molec-
ular architectures.[1] Optical imaging of intracellular molecules
is an arena where fluorogenic organic molecules play an indis-
pensable role in dissecting biological processes. The increasing
demand for photoluminescent materials for specific bioimag-
ing applications fueled the chemistry community to develop a
wealth of fluorescent dyes with enhanced photophysical prop-
erties. Given the spurring use of fluorescent microscopy in the
chemical biology field, it is worth noting that most widely
used fluorophores are based on approximately century-old
scaffolds (fluoresceins: 1871, coumarins; 1884, rhodamines:
1887, phenoxazines; 1903, cyanines: 1924),[2] and peripheral
structural manipulation of these established scaffolds is a com-
monly adopted maneuver for fine-tuning the properties.[3] Al-
A single-step protocol from readily available commercial chem-
icals, 2,5-diamino-4,6-dichloropyrimidine 1a and a variety of ar-
ylboronic acids forged the connectivity of the C4N4 core and
two aryl groups, allowing for the rapid production of a range
of intensely emissive compounds with distinct photophysical
properties. A generally large Stokes shift was observed, which
DFT studies revealed was due to structural relaxation to a
p-iminoquinone-like structure in the excited state. Additional
key features of C4N4 fluorophores (solid-state emissive; manip-
ulable at the two free amino groups; ON/OFF tunable by acyla-
tion of the 5-amino group; and positive solvatochromism)
make them a readily accessible new class of fluorophore with
modular synthesis capabilities that shares features of well-
qualified known fluorophores.[6] Fluorescent labeling of a pro-
tein and live-cell imaging demonstrate a promise in prospec-
tive bioimaging applications.
The ready availability of 4,6-dichloropyrimidines 1a–d with a
differential degree and pattern of the amino group appendage
enabled quick access to various 4,6-diphenylated pyrimidines
2a—d by Suzuki–Miyaura cross coupling (Figure 2a).[7] Unsym-
metrical derivative 3a-H was accessed by monophenylation
followed by a scission of the remaining CꢀCl bond under a hy-
drogen atmosphere. Analysis of the photophysical properties
of a series of these pyrimidine-based compounds revealed that
[a] Dr. H. Noda, Y. Asada, Dr. T. Maruyama, Dr. N. Takizawa, Dr. N. N. Noda,
Prof. Dr. M. Shibasaki, Dr. N. Kumagai
Institute of Microbial Chemistry (BIKAKEN), Tokyo
3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021 (Japan)
E-mail: mshibasa@bikaken.or.jp
nkumagai@bikaken.or.jp
The ORCID identification number(s) for the author(s) of this article can be
found under:
https://doi.org/10.1002/chem.201900467.
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Figure 2. a) Installation of phenyl groups on readily available chlorinated pyrimidines en route to fluorescent organic molecules. b) Schematic representation
of the pyrimidine derivatives with C4N[x]Ph[y] composition having different numbers of phenyl groups and nitrogen atoms, illuminating the significant
impact of 2,5-diamino- groups and 4,6-diphenyl- groups on photophysical properties. Wavelengths [nm] of absorption and emission maxima are shown. Ab-
solute quantum yield (excitation at 375 nm) was determined in CHCl3 by using a calibrated integrating sphere system. c) Photophysical properties of C4N4 de-
rivatives 2a and 7–11. [a] Photophysical data were taken from ref. [5a].
the photoluminescence emission was dependent on the posi-
tion and number of amino and phenyl groups on the pyrimi-
dine unit. Figure 2b displays the structures with photophysical
data as a function of the number of these peripheral substitu-
ents. Compound 2a, possessing the 2,5-diaminated C4N4 pyri-
midine unit, was an outlier in terms of its intensely emissive
nature (fF =0.56 in CHCl3) and large Stokes shift (112 nm). Un-
symmetrical 3a-H sharing the C4N4 unit but lacking one phenyl
group displayed a decreased quantum yield. A series of pyrimi-
dine compounds 2a–d possessing two phenyl groups at the
4,6-positions revealed that the 2,5-diamino group provided the
enhanced fluorescence intensity. Among two C4N3-type deriv-
atives, retention of the 5-amino group was of prime impor-
tance for radiative decay (2b) and deleting the 5-amino group
turned the fluorescence off (2c). As such, C4N3-type derivative
4 with an extra phenyl group at the 2-position had weak fluo-
rescence, whereas C4N2-type derivatives 2d and 5 lacking a 5-
amino group were virtually nonemissive. The lack of emission
from p-phenylenediamine derivative 6 underscored the re-
quirement for concurrent deployment of the pyrimidine unit
and 2,5-diamino groups to produce intense fluorescence.[8]
After having identified the diphenylated 2,5-diaminopyrimi-
dine possessing the C4N4 composition as a privileged fluoro-
genic scaffold, we performed a modular synthesis of C4N4 de-
rivatives 7–11 armed with a variety of 4,6-substituents via the
single-step procedure from 1a (Figure 2b). Figure 2c summa-
rizes the photophysical properties of six C4N4 derivatives 2a
and 7–11 in CHCl3 as well as in the solid state. Substituents at
the 4,6-positions dictated the maximum absorption and emis-
sion wavelength over the range of 100 nm with a generally
high absorption coefficient (f=0.8–1.4ꢁ104 mꢀ1 cmꢀ1), quan-
tum yield (fF =0.37–0.68), and large Stokes shift (mean
111 nm). Both electron-rich (8) and -deficient (10) aromatics af-
forded highly fluorescent materials.[9] To gain insight into the
photoluminescent properties as well as the origin of the large
Stokes shift of the C4N4 fluorophores, DFT calculations were
performed on prototype 2a using the Gaussian 16 package.[10]
Geometry optimization of the ground and excited states was
performed in the gas phase at the DFT/B3LYP/6–31+G(d) and
time-dependent DFT (TD-DFT)/CAM-B3LYP/6–31+G(d) level of
theory, respectively (Figure 3a).[11–13] The located ground-state
structure was characterized by a perfectly flat C4N4 core unit,
and the three key CꢀN bonds (C2ꢀN2, C4ꢀN3, and C5ꢀN5)
were consistent with those observed in the X-ray crystal struc-
ture (Tables S4 and S8, Supporting Information). In sharp con-
trast, significant deviation of the amino group at the 5-position
from the pyrimidine plane (19.98) was detected for the opti-
mized structure of the excited state with concomitant contrac-
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Figure 3. a) Calculated optimized structures of ground and excited state of 2a. Bond lengths are given in ꢂ. b) Electronic transition and Kohn–Sham frontier
orbitals of 2a by TD-DFT calculation at the CAM-B3LYP/6–311+ +G(2d,p)//B3LYP/6–31+G(d) level of theory (isovalue=0.02). f denotes oscillator strength
and energies are given in eV. c) Molecular packing of 2a in a single crystal viewed along the b axis. Head-to-head hydrogen bonding by the 2-aminopyrimi-
dine moiety is observed. d) Offset p interaction is detected between Ph and pyrimidine rings. e) Molecular packing of 9. p-Contact was not observed while
similar head-to-head hydrogen bonding is found. Color code; gray: carbon, blue: nitrogen. Hydrogen atoms are omitted for clarity.
tion of two CꢀN bonds (C2ꢀN2 and C5ꢀN5) on the periphery
of the pyrimidine ring and slight elongation of the C4ꢀN3
bond (Figure 3a, Table S4, Supporting Information). Together
with the apparent sp2 geometry of the two amino groups, the
excited state adopted a skewed p-iminoquinone-like structure,
which was consistent with the changes in the calculated
Wiberg bond index and charge distribution (Tables S5 and S6,
Supporting Information). The HOMOs and LUMOs of both the
ground and excited states are displayed in Figure 3b. While
both states have similar orbital distributions, comparison of
the HOMO and LUMO showed an apparent asymmetry; the
HOMO is mainly located at the central C4N4 unit and the
LUMO is delocalized over the peripheral two phenyl groups,
suggesting that vertical transition from S0!S1 is mainly attrib-
uted to a p!p* transition (Tables S1 and S2, Supporting Infor-
mation). Structural relaxation in the excited state characterized
by the p-iminoquinone-like structure and slight decrease in the
torsion angle of the phenyl group (42.6!32.48) lowers the
LUMO, but raises the HOMO level to a greater magnitude,
which corroborates the large Stokes shift of 2a.
nature of 2a, 7, and 11 in the solid state (fF = <0.03) (Fig-
ure 2c). The quantum yields of 8–10 in a solid state were con-
sistently lower than when in CHCl3, and AIE enhancement
(AIEE) was not observed.[15] X-ray crystallographic analysis of
nonemissive 2a and emissive 9 and 10 provided clues to the
diverted emission properties (Figure 3c–e). Although they
shared a primary local structure comprising one pair of mole-
cules via hydrogen bonding through the 2-amino pyrimidine
moiety (Figure 3c, e), these local structures are packed in a dis-
tinct manner. Packing of 2a is tighter (density 1.321 gcmꢀ3)
and each pyrimidine unit accepts p-contacts with a neighbor-
ing phenyl group (Figure 3d), suggesting that fluorescent
quenching occurs by rapid nonradiative energy transfer
through the Dexter mechanism (Figure 3c).[1a] Conversely, the
crystal structure of
9 is less densely packed (density
1.242 gcmꢀ3) and the m-terphenyl group effectively avoids in-
termolecular p-contact (Figure 3e), thereby producing pro-
nounced solid-state emission.
Only marginal differences in the solid-state emission wave-
length were detected compared with that in solution, indicat-
ing that structural relaxation upon excitation occurred under
the restricted mobility of the solid state similarly to that ob-
served in solution. Spectroscopic analysis of highly emissive 10
Compounds 8–10, bearing 3,5-disubstituted phenyl groups
exhibited intense solid-state emission spectra (fF =0.32, 0.37,
and 0.51, respectively), contrasting with the nonemissive
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(16), which could be exploited to develop a fluorescent probe
responsive to hydrolytic conditions.[17]
Next we turned our attention to bioimaging applications of
the C4N4 fluorophore.[18] Autophagy is a highly conserved in-
tracellular self-digestion process and garners increasing atten-
tion as its involvement is elucidated in a growing number of
biological events.[19] Accordingly, clarification of the interaction
between autophagy-related (Atg) proteins is of great impor-
tance to dissect this fundamental biological process. By har-
nessing the facile modification capabilities of the C4N4 fluoro-
phore, maleimide-armed derivative 17[20] was prepared and
subjected to conjugation with Atg8, a ubiquitin-like protein es-
sential for autophagy (Figure 6a).[21–23] Atg3 and Atg7 are
known as conjugating enzymes that produce Atg8 covalently
attached with phosphatidylethanolamine (Atg8-PE), whereas
deconjugating enzyme Atg4 cleaves Atg8-PE between the C-
terminus and the PE moiety.[24] Taking advantage of the shorter
excitation wavelength of 17, these three Atg enzymes were
differently labeled with longer-wavelength fluorophores to
Figure 4. a) Plot of emission wavenumber of 10 against the solvent polarity
(ET(30)) of aprotic solvents. Inset (upper right): itemized spectroscopic data
of 10 in aprotic solvent. Inset (lower left): itemized spectroscopic data of 10
in alcoholic solvent. cHex: cyclohexane, 1,4-diox: 1,4-dioxane.
in different media illuminated a two-phase trend. A clear posi-
tive solvatochromism was observed in aprotic solvents with a
high-fidelity linear relationship between the emission wave-
number and solvent polarity parameter ET(30) (Figure 4).[16]
A
small deviation in the wavelength of the absorption maxima
was detected compared with the fluorescence maxima, sug-
gesting only
a marginal difference between the dipole
moment of the equilibrium ground state and the Frank–
Condon excited state. In contrast, the photoluminescence be-
havior in protic solvents was largely uniform and the emission
wavelengths ranged from 535 to 540 nm, irrespective of ET(30)
(43.3–55.4), indicating that hydrogen-bonding interactions of
the C4N4 unit with solvent molecules determine the photolu-
minescence properties and override the dipole interactions.
Manipulation of the amino groups at the 2- and 5-positions
further supported the utility of the present C4N4-based fluoro-
phore (Figure 5). While dimethylation of the 2-amino group re-
sponded with a redshift of its absorption and emission band
(12), introduction of electron-withdrawing acyl groups resulted
in a significant blueshift (13). These derivatives maintained
emissive properties, implying that the 2-amino group of C4N4
fluorophores serves as a linking handle for prospective bio-
imaging applications. The 2-amino group could be replaced by
a methylsulfanyl group retaining a moderately emissive nature
(14), allowing for manipulation of the more hindered 5-amino
group (15, 16). In contrast to the 2-amino group, introducing
an acyl unit on the 5-amino group turned the fluorescence off
Figure 6. Confocal microphotographs of autophagy-related proteins and
HepG2 cells. a) Atg8, Atg3, Atg7, and Atg4 were labelled with 17, GFP,
SNAP-Surface 546, and SNAP-Surface Alexa Fluor 647, respectively, and excit-
ed at the specified wavelength. 6ꢁHis-fused Atg8 was bound on Ni-NTA
agarose resins. Cys-residue introduced at the C-terminus of Atg8 was modi-
fied for conjugation with 17. C147S mutant of Atg4 which loses deconjuga-
tion activity was used. b) Free fatty acid-treated HepG2 cells were incubated
with 10 (5 mm) and Nile red (1 mm) at 378C for 30 min before imaging. DIC:
differential interference contrast microscope.
Figure 5. Effects of substituents at the 2,5-positions of pyrimidine-based flu-
orophores on their spectroscopic properties in CHCl3.
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by fluorescent imaging. In the absence of Atg4, Atg3 and Atg7
made a complex with Atg8, whereas Atg4 dissociated the
Atg8–Atg3–Atg7 complex and solely interacted to Atg8. This
multicolour imaging illuminated a new finding that PE-conju-
gating enzymes (Atg3, Atg7) and PE-deconjugating enzyme
(Atg4) formed complexes with Atg8 in a mutually exclusive
fashion. The utility of the neutral C4N4 fluorophore was further
broadened by live-cell imaging (Figure 6b). Incubation of
seven C4N4 analogues with human hepatocellular carcinoma
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cytotoxic in the dark and under UV irradiation (365 nm,
2.5 Jcmꢀ2) with decent cell-membrane permeability.[25] Colocali-
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be manipulated by the installation of compartment-specific
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In conclusion, we developed a new fluorogenic scaffold
characterized by a pyrimidine-based core featuring a C4N4 ele-
mental composition. Two amino and aryl groups on the pyri-
midine core proved essential for intense emission. Generally,
large Stokes shifts and practical levels of quantum yields were
observed, which can be leveraged by facile single-step modu-
lar synthesis. The capability for protein labeling and live-cell
imaging support prospective utilities in bioimaging applica-
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Acknowledgements
This work was financially supported by KAKENHI (17H03025
and 18H04276 in Precisely Designed Catalysts with Customized
Scaffolding) from JSPS and MEXT. Dr. Tomoyuki Kimura is grate-
fully acknowledged for X-ray crystallographic analysis of 2a, 9,
and 10. We thank Dr. K. Matoba, Y. Ishii, and Y. Iwata for techni-
cal support in protein labeling and cell analysis. We are grate-
ful to Dr. R. Sawa, Y. Kubota, Dr. K. Iijima, and Y. Takahashi for
NMR and MS analyses.
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Conflict of interest
The Institute of Microbial Chemistry (BIKAKEN) has filed a
patent application for the use of C4N4 fluorophores as fluores-
cent materials.
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Keywords: bioconjugation · fluorescence · live-cell imaging ·
pyrimidine · Stokes shift
[18] Absorption and emission spectra of PEG- C4N4 as a model for bioconju-
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[25] See Supporting Information for details.
Manuscript received: January 30, 2019
Accepted manuscript online: February 4, 2019
Version of record online: && &&, 0000
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Chem. Eur. J. 2019, 25, 1 – 7
www.chemeurj.org
6
ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Communication
COMMUNICATION
A new scaffold for producing efficient
organic fluorescent materials was identi-
fied: 2,5-diamino-4,6-pyrimidine featur-
ing a C4N4 elemental composition (see
figure). Single-step installation of two
aryl groups at the 4,6-positions of the
pyrimidine core delivered fluorescent
organic materials in a modular fashion.
&
Fluorescence
H. Noda, Y. Asada, T. Maruyama,
N. Takizawa, N. N. Noda, M. Shibasaki,*
N. Kumagai*
&& – &&
A C4N4 Diaminopyrimidine
Fluorophore
Chem. Eur. J. 2019, 25, 1 – 7
www.chemeurj.org
7
ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ